The human T cell leukemia-lymphoma viruses (HTLV) are a family of related retroviruses originally isolated from patients with T cell lymphoma and cutaneous manifestations. A particular subgroup of the family, type I, also known as HTLV-I, is linked as causative agents of malignancies which share clinical and epidemiologic features with the disease called adult T-cell leukemia-lymphoma(ATL) that occurs in certain regions of Japan (6-9), the Caribbean Basin (10,11) and in southwestern United States (12).
Although the mechanism of transmission of HTLV-I is currently unknown, horizontal transmission of HTLV is clearly implicated by molecular and epidemiologic analyses (13,14). HTLV-I seropositivity in regions endemic for ATL is elevated overall in the general population and further elevated among close family members of patients and in the recipients of blood transfusions (15,16).
This means that there is an urgent need for a safe, reliable and sensitive test to screen each blood sample before its inclusion in blood banks to isolate blood samples which have been contaminated with HTLV-I virus to avoid the inadvertent spread of the virus among patients who must receive blood transfusions, e.g. hemophiliacs and surgical patients.
The complete nucleotide sequence of the HTLV-I virus has been reported in 1983 (17). This report elucidated the structure of the HTLV-I virus at both the DNA level and the predicted protein level and permits further serological studies of different epitopes which may be present on the HTLV-I virus.
Simultaneously, Dr. Carl Saxinger at National Cancer Institute reported the use of the isolated HTLV-I virus as a solid-phase immunoadsorbent for the development of an enzyme immunoassay for the detection of HTLV-I antibodies in the African population (18).
It is further reported by Samuel et al. (19) that a combined cloning and expression system in E. Coli has been used to identify HTLV-I DNA encoded glycoproteins which react immunologically with antibodies in sera from ATL patients. HTLV-I DNA encoding the envelope protein was cleaved into fragments and inserted into an expression vector. The expression vectors were introduced into an E. coli host by transformation. One clone, designates as pKS400, a envelope protein product was found to be suitable for use as an immunoadsorbent to screen a group of 28 coded sera. Antibodies that recognized the bacterially synthesized HTLV-I envelope protein sequences were found in all sera that had been shown to have antibodies to HTLV-I by an ELISA assay with disrupted virions as the antigen (18).
Slamon et al, Application No. PCT/US 85/01803, published on Mar. 27, 1986 under Publication No. W086/01834, described polypeptides associated with immunogenic sites of HTLV-I as expression products of the X region of HTLV-I, a highly conserved region located between env and the 3' LTR of the virus. The proteins have a molecular weight of between 37 kd and 40 kd and were cloned and expressed as fusion proteins in E. coli. The resulting products wee purified and used in liquid phase immunoprecipitation tests to screen sera. The results indicate an accuracy of from about 77% to 87%. (20)
Synthetic peptides have been used increasingly to map antigenic or immunogenic sites on the surface of proteins and for use as possible vaccines. We have previously taken this approach to identify and characterize highly antigenic epitopes on the envelope proteins of HTLV-III and developed sensitive and specific immunoassays for the detection of antibodies to HTLV-III (21) A similar approach is employed in this invention to select and identify highly antigenic epitopes in HTLV-I. In selecting regions of the envelope protein for epitope analysis, two strategies were applied. First, regions that exhibited a relatively high conversation of amino acid sequence between HTLV-I and HTLV-II were sought. Second, multiple overlapping linear peptides covering whole regions of gp21, the transmembrane portion of the HTLV-I envelope protein, were synthesized and characterized. Three peptides, with the following sequences, and a mixture thereof were found to be highly immunoreactive with sera from patients with ATL: EQU GLDLLFWEQGGLCKALQEQC-NH2 (I) EQU QNRRGLDLLFWEQGGLCKALQEQC-NH2 (II) EQU NRRGLDLLFWEQGGLC-NH2 (III)
wherein:
A=Ala=alanine, PA1 R=Arg=arginine, PA1 D=Asp=aspartic acid, PA1 G=Gly=glycine, PA1 I=Ile=isoleucine, PA1 F=Phe=phenylalanine, PA1 N=Asn=asparginine, PA1 Q=Gln=glutamine, PA1 E=Glu=glutamic acid, PA1 L=Leu=leucine, PA1 K=Lys=lysine, PA1 S=Ser=serine, PA1 W=Trp=tryptophan, PA1 Y=Tyr=tyrosine, PA1 V=Val=valine, PA1 C=Cys=cysteine. PA1 A=Ala;32 alanine, PA1 R=Arg=arginine, PA1 D=Asp=aspartic acid, PA1 N=Asn=asparagine, PA1 Q=Gln=glutamine, PA1 E=Glu=glutamic acid, PA1 L=Leu=leucine, PA1 K=Lys=lysine, PA1 G=Gly=glycine, PA1 I=Ile=isoleucine, PA1 F=Phe=phenylalanine, PA1 S=Ser=serine, PA1 W=Trp=trotophan, PA1 Y=Tyr=tyrosine, PA1 V=Val=valine, PA1 C=Cys=cysteine.
Assays for antibodies to HTLV-I based upon chemically synthesized peptides show several advantages over assays utilizing whole disrupted virus or bacterially produced immunoadsorbents. The peptides can easily be synthesized in gram quantities by using automated solid-phase methods, thus providing a reproducible antigen of high integrity with consistent yields. Isolation of antigens from biological systems precludes such reproducibility. More importantly, non-specific reactivities seen in non-HTLV-I infected individuals are likely due to the heterogeneity of the preparations used for assay. This is particularly true for assays using either the whole virus or Escherichia coli-derived recombinant products as immunoadsorbents. In these processes, the major histocompatibility antigens or endogenous bacterial proteins of the host cells are frequently copurified with the desired antigen virus or protein. Since antibodies to these contaminating antigens are frequently found in normal individuals, false-positive results cannot be eliminated by using current antigen isolation processes.
The assay of the present invention thus clearly eliminates the false-positive reactions encountered in the other methods and, at the same time, shows a high sensitivity to truly positive sera by the substantially increased signal-to-noise ratio. This increased signal-to-noise ratio likely results from the purity of the immunoadsorbent.
Furthermore, up to the present, no viable vaccine or method to provide protection against HTLV-I has been reported. The use of deactivated virus provokes fears of contracting the disease and would prevent its acceptability and use.
Similarly, the development of monoclonal and polyclonal antibodies to HTLV-I in mammals involves the use of HTLV-I as the immunogen and this presents unacceptable risks in the procedure.
It is, therefore, an objective of the present invention to develop a detection or diagnostic procedure that does not require the use of the virus or lysates thereof as a test reagent.
A further objective is to develop a test procedure that is highly sensitive and accurate.
Another objective is to develop a test that is highly sensitive so that very little test reagent or body fluid is needed to obtain an accurate result.
A further objective is to prepare a test reagent by chemical means. The synthetic reagent can then be used to detect the presence of antibodies to HTLV-I in body fluids and diagnose ATL, thereby avoiding the danger of exposure to the virus or segments thereof and the unnecessary proliferation of the virus.
Another objective is to develop a vaccine which when introduced into the body will stimulate production of antibodies to HTLV-I to provide protection against infection by HTLV-I in healthy mammals, including humans.
A further objective is to provide an immunogen which can be used for the development in mammals of monoclonal and polyclonal antibodies to HTLV-I which does not involve the use of HTLV-I as the immunogen.